Myeloid cell leukemia associated gene mcl-1

ABSTRACT

A gene, mcl-1, of the bcl-2 family is disclosed along with its nucleotide and amino acid sequence. Also disclosed are diagnostic and therapeutic methods of utilizing the mcl-1 nucleotide and polypeptide sequences.

This is a request for filing a divisional application of priorapplication Ser. No. 09/211,640, filed on Dec. 15, 1998, U.S. Pat. No.6,020,466, which is a division of application Ser. No. 08/441,375, filedon May 15, 1995, now U.S. Pat. No. 5,888,812, which is a division ofapplication Ser. No. 08/077,848, filed on Jun. 16, 1993 and issued intoU.S. Pat. No. 5,470,955, which is a continuation-in-part application ofSer. No. 08/012,307, filed Feb. 2, 1993, abandoned.

This invention was made with Government support under Grant No. CA54385awarded by the National Institutes of Health. The Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to unique proto-oncogenepolypeptides and specifically to a novel polypeptide of the bcl-2 familyand its nucleic acid sequence.

2. Description of the Related Art

Advances in recombinant DNA technology have led to the discovery ofnormal cellular genes (proto-oncogenes and tumor suppressor genes, andapoptosis/cell death-related genes) which control growth, development,and differentiation. Under certain circumstances, regulation of thesegenes is altered and normal cells assume neoplastic growth behavior. Insome cases, the normal cell phenotype can be restored by variousmanipulations associated with these genes. There are over 40 knownproto-oncogenes and suppressor genes to date, which fall into variouscategories depending on their functional characteristics. Theseinclude, 1) growth factors and growth factor receptors, 2) messengers ofintracellular signal transduction pathways, for example, between thecytoplasm and the nucleus, and 3) regulatory proteins influencing geneexpression and DNA replication.

Qualitative changes in the structure of proto-oncogenes or theirproducts and quantitative changes in their expression have beendocumented for several cancers. With chronic myelogenous leukemia, forexample, the abl oncogene is translocated to chromosome 22 in thevicinity of the bcr gene. A cancer specific fusion protein,qualitatively different from parent cell proteins, is produced and is anideal cancer marker. Mutant ras genes have been implicated in theearliest stages of human leukemias and colon cancers. The detection ofthese mutations in defined premalignant states could provide valuableprognostic information for clinicians.

During their life span, cells normally pass from an immature state withproliferative potential, through sequential stages of differentiation,to eventual cell death. This orderly progression is aberrant in cancer,probably due to alterations in oncogenes, tumor suppressor genes, andother genes. The progression from the immature state to differentiationcan be reestablished in inducible leukemia cell lines. For example, ML-1human myeloblastic leukemia cells can be induced to differentiate tomonocytes/macrophages with the phorbol ester,12-O-tetradecanoylphorbol-13-acetate (TPA). The differentiated cellslose proliferative capacity and accumulate in the G₀/G₁ phase of thecell cycle, while remaining viable and capable of carrying out normalmonocyte/macrophage functions. In general, immature, proliferative cellsconvert to a differentiated, viable, non-proliferative phenotype.

In ML-1 cells, the initial induction of “programming” of this conversioncan be separated from the subsequent phenotypic changes. When cells areinduced with TPA for three hours under specific conditions, they becomeirreversibly committed to undergo differentiation over the next threedays. This temporal separation can be used to identify genes thatincrease in expression during the early programming of differentiation.Such “early-induction” genes might influence or help bring about thelater phenotypic conversion. Aberrant expression of theseearly-induction genes, such as the proto-oncogene fos, may lead todevelopment of a transformed phenotype.

Research on oncogenes and their products is motivated partly by thebelief that a more fundamental understanding of the mechanisms of cancercausation and maintenance will lead to more rational means of diagnosingand treating malignancies. Using family studies of restriction fragmentlength polymorphisms (RFLPs) genetically linked to proto-oncogenes, itmay be possible to identify cancer-prone individuals.

Current cancer tests are nonspecific and of limited clinicalapplication. For example, a biochemical test, widely used for bothdiagnostic and monitoring of cancer, measures level of carcinoembryonicantigen (CEA). CEA is an oncofetal antigen detectable in large amountsin embryonal tissue, but in small amounts in normal adult tissues. Serumof patients with certain gastrointestinal cancers contains elevated CEAlevels that can be measured by immunological methods. The amount of CEAin serum correlates with the remission or relapse of these tumors, withthe levels decreasing abruptly after surgical removal of the tumor. Thereturn of elevated CEA levels signifies a return of malignant cells.CEA, however, is also a normal glycoprotein found at low levels innearly all adults. Moreover, this protein can be elevated with severalnonmalignant conditions and is not elevated in the presence of manycancers. Therefore, it is far from ideal as a cancer marker.

A similar oncofetal tumor marker is alpha-fetoprotein, an embryonic formof albumin. Again, the antigen is detectable in high amounts inembryonal tissue and in low amounts in normal adults. It is elevated ina number of gastrointestinal malignancies including hepatoma. Like CEA,a decrease correlates with the remission of cancer and a re-elevationwith relapse. There is insufficient sensitivity and specificity to makethis marker useful for screening for malignancy or for monitoringpreviously diagnosed cancer in any but a few selected cases.

For years, various therapeutic agents have been used to alter theexpression of genes or the translation of their messages into proteinproducts. However, a major problem with these agents is that they tendto act indiscriminately such that healthy cells as well as malignantcells are affected. As a consequence existing chemotherapeutic regimesare often associated with severe side effects due to the non-specificactivity of these agents.

One possible approach to specific intentional therapy is by targetingcells expressing particular oncogenes, tumor suppressor genes orapoptosis/cell death genes. Therefore, there is a continual need toidentify new oncogenes associated with cancer and neoplastic phenotypesand with the suppression of these phenotypes. Once these genes areidentified, specific therapeutics may be designed which are directed,for example, against the genes themselves, their RNA transcripts ortheir protein products which should have minimal detrimental effect onhealthy cells.

SUMMARY OF THE INVENTION

The present invention arose from the seminal discovery of a new gene,mcl-1, which is associated with certain cell proliferative disorders.This new gene was initially identified based on expression during theprogramming of differentiation in myeloid cell leukemia. As a result ofthis pioneering discovery, the present invention provides at its mostfundamental level, a functional polypeptide, mcl-1, and thepolynucleotide which encodes mcl-1. The novel polypeptide allows theproduction of antibodies which are immunoreactive with all or a portionof mcl-1, which can be utilized in various diagnostic and therapeuticmodalities to detect and treat cell proliferative disorders associatedwith mcl-1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show a time course of expression of mcl-1 during theTPA-induced differentiation of ML-1 cells.

FIG. 2A-B show the deduced amino acid sequence of the mcl-1 protein andschematic representation of the cDNA.

FIG. 3 shows in vitro translation of mcl-1 mRNA.

FIG. 4 shows the amino acid alignment of the carboxyl regions of mcl-1,bcl-2, and BHRF1.

FIGS. 5A-5B are the nucleotide sequence of mcl-1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel polypeptide, mcl-1, which isexpressed early during the programming of differentiation of myeloidcell leukemia. Genes expressed early in cell differentiation mayparticipate in the induction or programming of the ensuing phenotypicchanges. Also included is the polynucleotide sequence which encodesmcl-1 or portions thereof. The carboxyl portion of mcl-1 has homology tobcl-2, which inhibits programmed cell death in developing lymphoid cellsand lymphoma. The mcl-1/bcl-2 family of genes are identified in cancercells, but are distinct from known oncogenes in that they arecharacterized by an association with the programming of transitions incell fate, such as from viability to death or from proliferation todifferentiation. The invention provides a 3946 base pair polynucleotidewhich encodes a 37.5 kD polypeptide of the bcl-2 family. The inventionalso includes antibodies immunoreactive with mcl-1 polypeptide orfragments of the polypeptide. The invention also provides a method foridentifying a cell expressing mcl-1 and a method for treating an mcl-1associated disorder.

As used herein, the term “functional polypeptide” refers to apolypeptide which possesses a biological function or activity which isidentified through a defined functional assay and which is associatedwith a particular biologic, morphologic or phenotypic alteration in thecell. The biological function can vary from a polypeptide fragment assmall as an epitope to which an antibody molecule can bind to as largeas a polypeptide which is capable of participating in the characteristicinduction or programming of phenotypic changes within a cell. A“functional polynucleotide” denotes a polynucleotide which encodes afunctional polypeptide as described herein.

The term “substantially pure” means any mcl-1 polypeptide of the presentinvention, or any gene encoding an mcl-1 polypeptide, which isessentially free of other polypeptides or genes, respectively, or ofother contaminants with which it might normally be found in nature, andas such exists in a form not found in nature. By “functional derivative”is meant the “fragments,” “variants,” “analogues,” or “chemicalderivatives” of a molecule. A “fragment” of a molecule, such as any ofthe DNA sequences of the present invention, includes any nucleotidesubset of the molecule. A “variant” of such molecule refers to anaturally occurring molecule substantially similar to either the entiremolecule, or a fragment thereof. An “analog” of a molecule refers to anon-natural molecule substantially similar to either the entire moleculeor a fragment thereof.

A molecule is said to be “substantially similar” to another molecule ifthe sequence of amino acids in both molecules is substantially the same.Substantially similar amino acid molecules will possess a similarbiological activity. Thus, provided that two molecules possess a similaractivity, they are considered variants as that term is used herein evenif one of the molecules contains additional amino acid residues notfound in the other, or if the sequence of amino acid residues is notidentical. As used herein, a molecule is said to be a “chemicalderivative” of another molecule when it contains additional chemicalmoieties not normally a part of the molecule. Such moieties may improvethe molecule's solubility, absorption, biological half life, etc. Themoieties may alternatively decrease the toxicity of the molecule,eliminate or attenuate any undesirable side effect of the molecule, etc.Moieties capable of mediating such effects are disclosed, for example,in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co.,Easton, Pa. (1980).

Similarly, a “functional derivative” of a gene encoding mcl-1polypeptide of the present invention includes “fragments,” “variants”,or “analogues” of the gene, which may be “substantially similar” innucleotide sequence, and which encode a molecule possessing similaractivity to mcl-1 peptide.

Thus, as used herein, mcl-1 polypeptide includes any functionalderivative, fragments, variants, analogues, chemical derivatives whichmay be substantially similar to the mcl-1 polypeptide described hereinand which possess similar activity.

Minor modifications of the mcl-1 primary amino acid sequence may resultin proteins which have substantially equivalent activity as compared tothe mcl-1 polypeptide described herein. Such modifications may bedeliberate, as by site-directed mutagenesis, or may a spontaneous. Allof the polypeptides produced by these modifications are included hereinas long as the biological activity of mcl-1 still exists. Further,deletion of one or more amino acids can also result in a modification ofthe structure of the resultant molecule without significantly alteringits biological activity. This can lead to the development of a smalleractive molecule which would have broader utility. For example, one canremove amino or carboxy terminal amino acids which may not be requiredfor mcl-1 biological activity.

The term “conservative variation” as used herein denotes the replacementof an amino acid residue by another, biologically similar residue.Examples of conservative variations include the substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas the substitution of arginine for lysine, glutamic for aspartic acids,or glutamine for asparagine, and the like. The term “conservativevariation” also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid provided that antibodies raised tothe substituted polypeptide also immunoreact with the unsubstitutedpolypeptide.

Peptides of the invention can be synthesized by the well known solidphase peptide synthesis methods described Merrifield, J. Am. Chem. Soc.,85:2149, 1962), and Stewart and Young, Solid Phase Peptides Synthesis,(Freeman, San Francisco, 1969, pp. 27-62), using acopoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer.On completion of chemical synthesis, the peptides can be deprotected andcleaved from the polymer by treatment with liquid HF-10% anisole forabout ¼-1 hours at 0° C. After evaporation of the reagents, the peptidesare extracted from the polymer with 1% acetic acid solution which isthen lyophilized to yield the crude material. This can normally bepurified by such techniques as gel filtration on Sephadex G-15 using 5%acetic acid as a solvent. Lyophilization of appropriate fractions of thecolumn will yield the homogeneous peptide or peptide derivatives, whichcan then be characterized by such standard techniques as amino acidanalysis, thin layer chromatography, high performance liquidchromatography, ultraviolet absorption spectroscopy, molar rotation,solubility, and quantitated by the solid phase Edman degradation.

As used herein, the terms “polynucleotide” or “mcl-1 polynucleotide”denotes DNA, cDNA and RNA which encode mcl-1 polypeptide as well asuntranslated sequences which flank the structural gene encoding mcl-1.It is understood that all polynucleotides encoding all or a portion ofmcl-1 polypeptide of the invention are also included herein, as long asthe encoded polypeptide exhibits the activity or function of mcl-1 orthe tissue expression pattern characteristic of mcl-1. Suchpolynucleotides include naturally occurring forms, such as allelicvariants, and intentionally manipulated forms, for example, mutagenizedpolynucleotides, as well as artificially synthesized polynucleotides.Such mutagenized polynucleotides can be produced, for example, bysubjecting mcl-1 polynucleotide to site-directed mutagenesis.

As described above, in another embodiment, a polynucleotide of theinvention also includes in addition to mcl-1 coding regions, thosenucleotides which flank the coding region of the mcl-1 structural gene.For example, a polynucleotide of the invention includes 5′ regulatorynucleotide sequences and 3′ untranslated sequences associated with themcl-1 structural gene. Analogous to bcl-2 (Cotter, et al., Blood,76:131, 1990), oligonucleotide primers such as those representingnucleotide sequences in the major breakpoint region (mbr) or the minorcluster region (mcr) which flank a translocation region are useful inthe polymerase chain reaction (PCR) for amplifying and detectingtranslocations associated with the mcl-1 gene. The primers may representuntranslated nucleotide sequences which detect sequence junctionsproduced by translocation in various mcl-1 associated cell proliferativedisorders, for example.

The polynucleotide sequence for mcl-1 also includes antisense sequences.The polynucleotides of the invention also include sequences that aredegenerate as a result of the genetic code. There are 20 natural aminoacids, most of which are specified by more than one codon. Therefore, aslong as th amino acid sequence of mcl-1 results in a functionalpolypeptide (at least, in the case of the sense polynucleotide strand),all degenerate nucleotide sequences are included in the invention. Wherethe antisense polynucleotide is concerned, the invention embraces allantisense polynucleotides capable of inhibiting production of mcl-1polypeptide.

The preferred mcl-1 cDNA clone of the invention is defined by a sequenceof 3946 basepairs, in accord with the longest transcript of 3.8 kb. Thepreferred mcl-1 encoded protein is approximately 350 amino acids and hasa molecular weight of approximately 37.5 kD. In its amino terminalportion, the mcl-1 protein contains two “PEST” sequences, enriched inproline (P), glutamic acid (E), serine (S), and threonine (T) and fourpairs of arginines. “PEST” sequences are present in a variety ofoncoproteins and other proteins that undergo rapid turn-over. These“PEST” sequences are not found in the bcl-2 encoding polynucleotidesequence and, thus, represent a characteristic feature of members of themcl-1 polypeptide family. It is in the carboxyl region that mcl-1 hassequence homology to bcl-2 (35% amino acid identity and 59% similarityin 139 amino acid residues).

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization procedures whichare well known in the art. These include, but are not limited to: 1)hybridization of probes to genomic or cDNA libraries to detect sharednucleotide sequences; 2) antibody screening of expression libraries todetect shared structural features and 3) synthesis by the polymerasechain reaction (PCR).

Hybridization procedures are useful for the screening of recombinantclones by using labeled mixed synthetic oligonucleotide probes whereeach probe is potentially the complete complement of a specific DNAsequence in the hybridization sample which includes a heterogeneousmixture of denatured double-stranded DNA. For such screening,hybridization is preferably performed on either single-stranded DNA ordenatured double-stranded DNA. Hybridization is particularly useful inthe detection of cDNA clones derived from sources where an extremely lowamount of mRNA sequences relating to the polypeptide of interest arepresent. In other words, by using stringent hybridization conditionsdirected to avoid non-specific binding, it is possible, for example, toallow the autoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace, et al., Nucleic AcidResearch, 9:879, 1981).

A mcl-1 containing cDNA library can be screened by injecting the variouscDNAs into oocytes, allowing sufficient time for expression of the cDNAgene products to occur, and testing for the presence of the desired cDNAexpression product, for example, by using antibody specific for mcl-1polypeptide or by using functional assays for mcl-1 activity and atissue expression pattern characteristic of mcl-1. alternatively, a cDNAlibrary can be screened indirectly for mcl-1 polypeptides having atleast one epitope using antibodies specific for mcl-1. Such antibodiescan be either polyclonally or monoclonally derived and used to detectexpression product indicative of the presence of mcl-1 cDNA.

Screening procedures which rely on nucleic acid hybridization make ispossible to isolate any gene sequence from any organism, provided theappropriate probe is available. Oligonucleotide probes, which correspondto a part of the sequence encoding the protein in question, can besynthesized chemically. This require that short, oligopeptide stretchesof amino acid sequence must be known. The DNA sequence encoding theprotein can be deduced from the genetic code, however, the degeneracy ofthe code must be taken into account. It is possible to perform a mixedaddition reaction when the sequence is degenerate. This includes aheterogeneous mixture of denatured double-stranded DNA. For suchscreening, hybridization is preferably performed on eithersingle-stranded DNA or denatured double-stranded DNA. Hybridization isparticularly useful in the detection of cDNA clones derived from sourceswhere an extremely low amount of mRNA sequences relating to thepolypeptide of interest are present. In other words, by using stringenthybridization conditions directed to avoid non-specific binding, it ispossible, for example, to allow the autoradiographic visualization of aspecific cDNA clone by the hybridization of the target DNA to thatsingle probe in the mixture which is its complete complement (Wallace,et al., Nucl. Acid Res., 9:879, 1981).

The development of specific DNA sequences encoding mcl-1 can also beobtained by: 1) isolation of double-stranded DNA sequences from thegenomic DNA; 2) chemical manufacture of a DNA sequence to provide thenecessary codons for the polypeptide of interest; and 3) in vitrosynthesis of a double-stranded DNA sequence by reverse transcription ofmRNA isolated from a eukaryotic donor cell. In the latter case, adouble-stranded DNA complement of mRNA is eventually formed which isgenerally referred to as cDNA. Of these three methods for developingspecific DNA sequences for use in recombinant procedures, the isolationof genomic DNA isolates is the least common. This is especially truewhen it is desirable to obtain the microbial expression of mammalianpolypeptides due to the presence of introns.

The synthesis of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptide is not known, the direct synthesis of DNA sequencesis not possible and the method of choice is the synthesis of cDNAsequences. Among the standard procedures for isolating cDNA sequences ofinterest is the formation of plasmid- or phage-carrying cDNA librarieswhich are derived from reverse transcription of mRNA which is abundantin donor cells that have a high level of genetic expression. When usedin combination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have bee denatured into asingle-stranded form (Jay, et al., Nucl. Acid Res., 11:2325, 1983).

A cDNA expression library, such as lambda gt11, can be screenedindirectly for mcl-1 peptides having at lest one epitope, usingantibodies specific for mcl-1. Such antibodies can be eitherpolyclonally or monoclonally derived and used to detect expressionproduct indicative of the presence of mcl-1 cDNA. DNA sequences encodingmcl-1 can be expressed in vitro by DNA transfer into a suitable hostcell. “Host cells” are cells in which a vector can be propagated and itsDNA expressed. The term also includes any progeny of the subject hostcell. It is understood that all progeny may not be identical to theparental cell since there may be mutations that occur duringreplication. However, such progeny are included when the term “hostcell” is used. Methods of stable transfer, in other words when theforeign DNA is continuously maintained in the host, are known in theart.

In the present invention, the mcl-1 polynucleotide sequences may beinserted into a recombinant expression vector. The term “recombinantexpression vector” refers to a plasmid, virus or other vehicle known inthe art that has been manipulated by insertion or incorporation of themcl-1 genetic sequences. Such expression vectors contain a promotersequence which facilitates the efficient transcription of the insertedgenetic sequence of the host. The expression vector typically containsan origin of replication, a promoter, as well as specific genes whichallow phenotypic selection of the transformed cells. Vectors suitablefor use in the present invention include, but are not limited to theT7-based expression vector for expression in bacteria (Rosenberg, etal., Gene, 56:125, 1987), the pMSXND expression vector for expression inmammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, metallothionein I, orpolyhedrin promoters).

Polynucleotide sequences encoding mcl-1 can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art. Such vectorsare used to incorporate DNA sequences of the invention.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ method byprocedures well known in the art. Alternatively, MgCl₂ or RbCl can beused. Transformation can also be performed after forming a protoplast ofthe host cell or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the mcl-1 of the invention,and a second foreign DNA molecule encoding a selectable phenotype, suchas the herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein. (Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982).

Isolation and purification of microbial expressed polypeptide, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

The invention includes polyclonal and monoclonal antibodiesimmunoreactive with mcl-1 polypeptide or immunogenic fragments thereof.If desired, polyclonal antibodies can be further purified, for example,by binding to and elution from a matrix to which mcl-1 polypeptide isbound. Those of skill in the art will know of various other techniquescommon in the immunology arts for purification and/or concentration ofpolyclonal antibodies, as well as monoclonal antibodies. Antibody whichconsists essentially of pooled monoclonal antibodies with differentepitopic specificities, as well as distinct monoclonal antibodypreparations are provided. Monoclonal antibodies are made from antigencontaining fragments of the protein by methods well known to thoseskilled in the art (Kohler, et al., Nature, 256:495, 1975). The termantibody or, immunoglobulin as used in this invention includes intactmolecules as well as fragments thereof, such as Fab and F(ab′)₂, whichare capable of binding an epitopic determinant on mcl-1.

A preferred method for the identification and isolation of antibodybinding domain which exhibit binding with mcl-1 is the bacteriophage λvector system. This factor system has been used to express acombinatorial library of Fab fragments from the mouse antibodyrepertoire in Escherichia coli (Huse, et al., Science, 246:1275-1281,1989) and from the human antibody repertoire (Mullinax, et al., Proc.Natl. Acad. Sci., 87:8095-8099, 1990). As described therein, receptors(Fab molecules) exhibiting binding for a preselected ligand wereidentified and isolated from these antibody expression libraries. Thismethodology can also be applied to hybridoma cell lines expressingmonoclonal antibodies with binding for a preselected ligand. Hybridomaswhich secrete a desired monoclonal antibody can be produced in variousways using techniques well understood by those having ordinary skill inthe art and will not be repeated here. Details of these techniques aredescribed in such references as Monoclonal Antibodies-Hybridomas: A NewDimension in Biological Analysis, Edited by Roger H. Kennett, et al.,Plenum Press, 1980; and U.S. Pat. No. 4,172,124.

The term “cell-proliferative disorder” denotes malignant as well asnon-malignant cell populations which often appear to differ from thesurrounding tissue both morphologically and genotypically. Suchdisorders may be associated, for example, with abnormal expression ofmcl-1. “Abnormal expression” encompasses both increased or decreasedlevels of expression of mcl-1, as well as expression of a mutant form ofmcl-1 such that the normal function of mcl-1 is altered. Abnormalexpression also includes inappropriate expression of mcl-1 during thecell cycle or in an incorrect cell type. The mcl-1 polynucleotide in theform of an antisense polynucleotide is useful in treating malignanciesof the various organ systems, particularly, for example, those oflymphoid origin such as lymphoma. Essentially, any disorder which isetiologically linked to altered expression of mcl-1 could be consideredsusceptible to treatment with a reagent of the invention which modulatesmcl-1 expression. The term “modulate” envisions the suppression ofexpression of mcl-1 when it is over-expressed, or augmentation of mcl-1expression when it is under-expressed or when the mcl-1 expressed is amutant form of the polypeptide. When a cell proliferative disorder isassociated with mcl-1 overexpression, such suppressive reagents asantisense mcl-1 polynucleotide sequence or mcl-1 binding antibody can beintroduced to a cell. Alternatively, when a cell proliferative disorderis associated with underexpression or expression of a mutant mcl-1polypeptide, a sense polynucleotide sequence (the DNA coding strand) ormcl-1 polypeptide can be introduced into the cell.

The invention provides a method for detecting a cell expressing mcl-1 ora cell proliferative disorder associated with mcl-1 comprisingcontacting a cell suspected of expressing mcl-1 or having a mcl-1associated disorder, with a reagent which binds to the component. Thecell component can be nucleic acid, such as DNA or RNA, or protein. Whenthe component is nucleic acid, the reagent is a nucleic acid probe orPCR primer. When the cell component is protein, the reagent is anantibody probe. The probes are detectably labeled, for example, with aradioisotope, a fluorescent compound, a bioluminescent compound, achemiluminescent compound, a metal chelator or an enzyme. Those ofordinary skill in the art will know of other suitable labels for bindingto the antibody, or will be able to ascertain such, using routineexperimentation.

For purposes of the invention, an antibody or nucleic acid probespecific for mcl-1 may be used to detect the presence of mcl-1polypeptide (using antibody) or polynucleotide (using nucleic acidprobe) in biological fluids or tissues. The use of oligonucleotideprimers based on translocation regions in the mcl-1 sequence are usefulfor amplifying DNA, for example by PCR, and analysis of thetranslocation junctions. Any specimen containing a detectable amount ofantigen can be used. A preferred sample of this invention is tissue oflymphoid origin, specifically tissue containing hematopoietic cells.More specifically, the hematopoietic cells are preferably myeloid cells.Preferably the subject is human.

Another technique which may also result in greater sensitivity consistsof coupling the antibodies to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin, which reactswith avidin, or dinitrophenyl, pyridoxal, and fluorescein, which canreact with specific anti-hapten antibodies.

The method for detecting a cell expressing mcl-1 or a cell proliferativedisorder associated with mcl-1, described above, can be utilized fordetection of residual myeloid leukemia or other cells in a subject in astate of clinical remission. Additionally, the method for detectingmcl-1 polypeptide in cells is useful for detecting a cell proliferativedisorder by identifying cells expressing mcl-1 at levels different thannormal cells. Using the method of the invention, high, low, and mutantmcl-1 expression can be identified in a cell and the appropriate courseof treatment can be employed (e.g., sense or antisense gene therapy).

The monoclonal antibodies of the invention are suited for use, forexample, in immunoassays in which they can be utilized in liquid phaseor bound to a solid phase carrier. In addition, the monoclonalantibodies in these immunoassays can be detectably labeled in variousways. Examples of types of immunoassays which can utilize monoclonalantibodies of the invention are competitive and non-competitiveimmunoassays in either a direct or indirect format. Examples of suchimmunoassays are the radioimmunoassay (RIA) and the sandwich(immunometric) assay. Detection of the antigens using the monoclonalantibodies of the invention can be done utilizing immunoassays which arerun in either the forward, reverse, or simultaneous modes, includingimmunohistochemical assays on physiological samples. Those of skill inthe art will know, or can readily discern, other immunoassay formatswithout undue experimentation.

The monoclonal antibodies of the invention can be bound to manydifferent carriers and used to detect the presence of mcl-1. Examples ofwell-known carriers include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses and magnetite. The nature of the carrier canbe either soluble or insoluble for purposes of the invention. Thoseskilled in the art will know of other suitable carriers for bindingmonoclonal antibodies, or will be able to ascertain such using routineexperimentation.

For purposes of the invention, mcl-1 may be detected by the monoclonalantibodies of the invention when present in biological fluids andtissues. Any sample containing a detectable amount of mcl-1 can be used.A sample can be a liquid such as urine, saliva, cerebrospinal fluid,blood, serum and the like, or a solid or semi-solid such as tissues,feces, and the like, or, alternatively, a solid tissue such as thosecommonly used in histological diagnosis.

As used in this invention, the term “epitope” includes any determinantcapable of specific interaction with the monoclonal antibodies of theinvention. Epitopic determinants usually consist of chemically activesurface groups of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

In using the monoclonal antibodies of the invention for the in vivodetection of antigen, the detectably labeled monoclonal antibody isgiven in a dose which is diagnostically effective. The term“diagnostically effective” means that the amount of detectably labeledmonoclonal antibody is administered in sufficient quantity to enabledetection of the site having the mcl-1 antigen for which the monoclonalantibodies are specific.

The concentration of detectably labeled monoclonal antibody which isadministered should be sufficient such that the binding to those cellshaving mcl-1 is detectable compared to the background. Further, it isdesirable that the detectably labeled monoclonal antibody be rapidlycleared from the circulatory system in order to give the besttarget-to-background signal ratio.

As a rule, the dosage of detectably labeled monoclonal antibody for invivo diagnosis will vary depending on such factors as age, sex, andextent of disease of the individual. The dosage of monoclonal antibodycan vary from about 0.001 mg/m² to about 500 mg/m², preferably 0.1 mg/m²to about 200 mg/m², most preferably about 0.1 mg/m² to about 10 mg/m².Such dosages may vary, for example, depending on whether multipleinjections are given, tumor burden, and other factors known to those ofskill in the art.

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that the half-life of theradioisotope be long enough so that it is still detectable at the timeof maximum uptake by the target, but short enough so that deleteriousradiation with respect to the host is minimized. Ideally, a radioisotopeused for in vivo imaging will lack a particle emission, but produce alarge number of photons in the 140-250 keV range, which may be readilydetected by conventional gamma cameras.

For in vivo diagnosis, radioisotopes may be bound to immunoglobulineither directly or indirectly by using an intermediate functional group.Intermediate functional groups which often are used to bindradioisotopes which exist as metallic ions to immunoglobulins are thebifunctional chelating agents such as diethylenetriaminepentacetic acid(DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.Typical examples of metallic ions which can be bound to the monoclonalantibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, and²⁰¹TI.

The monoclonal antibodies of the invention can also be labeled with aparamagnetic isotope for purposes of in vivo diagnosis, as in magneticresonance imaging (MRI) or electron spin resonance (ESR). In general,any conventional method for visualizing diagnostic imaging can beutilized. Usually gamma and positron emitting radioisotopes are used forcamera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹³⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr,and ⁵⁶Fe.

The monoclonal antibodies of the invention can be used to monitor thecourse of amelioration of mcl-1 associated cell proliferative disorder.Thus, by measuring the increase or decrease in the number of cellsexpressing mcl-1 or changes in the concentration of normal versus mutantmcl-1 present in various body fluids, it would be possible to determinewhether a particular therapeutic regiment aimed at ameliorating thedisorder is effective.

The present invention also provides a method for treating a subject witha mcl-1 associated cell proliferative disorder. The mcl-1 nucleotidesequence can be expressed in an altered manner as compared to expressionin a normal cell, therefore it is possible to design appropriatetherapeutic or diagnostic techniques directed to this sequence. Thus,where a cell-proliferative disorder is associated with theover-expression of mcl-1, nucleic acid sequences that interfere withmcl-1 expression at the translational level can be used. This approachutilizes, for example, antisense nucleic acid and ribozymes to blocktranslation of a specific mcl-1 mRNA, either by masking that mRNA withan antisense nucleic acid or by cleaving it with a ribozyme. In caseswhen a cell proliferative disorder or abnormal cell phenotype isassociated with the under expression of mcl-1 or expression of a mutantmcl-1 polypeptide, nucleic acid sequences encoding mcl-1 (sense) couldbe administered to the subject with the disorder.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub, ScientificAmerican, 262:40, 1990). In the cell, the antisense nucleic acidshybridize to the corresponding mRNA, forming a double-stranded molecule.The antisense nucleic acids interfere with the translation of the mRNAsince the cell will not translate a mRNA that is double-stranded.Antisense oligomers of about 15 nucleotides are preferred, since theyare easily synthesized and are less likely to cause problems than largermolecules when introduced into the target mcl-1-producing cell. The useof antisense methods to inhibit the in vitro translation of genes iswell known in the art (Marcus-Sakura, Anal. Biochem., 172:289, 1988).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that that sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

The present invention also provides gene therapy for the treatment ofcell proliferative disorders which are mediated by mcl-1 protein. Suchtherapy would achieve its therapeutic effect by introduction of themcl-1 antisense polynucleotide, into cells of subjects having theproliferative disorder. Delivery of antisense mcl-1 polynucleotide canbe achieved using a recombinant expression vector such as a chimericvirus or a colloidal dispersion system. Disorders associated withunder-expression of mcl-1 could similarly be treated using gene therapywith sense nucleotide sequences.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. By inserting a mcl-1 sequence of interestinto the viral vector, along with another gene which encodes the ligandfor a receptor on a specific target cell, for example, the vector is nowtarget specific. Retroviral vectors can be made target specific byinserting, for example, a polynucleotide encoding a sugar, a glycolipid,or a protein. Preferred targeting is accomplished by using an antibodyto target the retroviral vector. Those of skill in the art will knownof, or can readily ascertain without undue experimentation, specificpolynucleotide sequences which can be inserted into the retroviralgenome to allow target specific delivery of the retroviral vectorcontaining the mcl-1 antisense polynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsidation. Helper cell lines which havedeletions of the packaging signal include but are not limited to Ψ2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced.

Alternatively, NIH 3T3 or other tissue culture cells can be directlytransfected with plasmids encoding the retroviral structural genes gag,pol and env, by conventional calcium phosphate transfection. These cellsare then transfected with the vector plasmid containing the genes ofinterest. The resulting cells release the retroviral vector into theculture medium.

Another targeted delivery system for mcl-1 antisense polynucleotides acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is liposome. Liposomes are artificial membrane vesicles whichare useful as delivery vehicles in vitro and in vivo. It has been shownthat large unilamellar vesicles (LUV), which range in size from 0.2-4.0um can encapsulate a substantial percentage of an aqueous buffercontaining large macromolecules. RNA, DNA and intact virions can beencapsulated within the aqueous interior and be delivered to cells in abiologically active form (Fraley, et al., Trends Biochem. Sci., 6:77,1981). In addition to mammalian cells, liposomes have been used fordelivery of polynucleotides in plant, yeast and bacterial cells. Inorder for a liposome to be an efficient gene transfer vehicle, thefollowing characteristics should be present: (1) encapsulation of thegenes of interest at high efficiency while not compromising theirbiological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes has been classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectively, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

In general, the compounds bound to the surface of the targeted deliverysystem will be ligands and receptors which will allow the targeteddelivery system to find and “home in” on the desired cells. A ligand maybe any compound of interest which will bind to another compound, such asa receptor.

In general, surface membrane proteins which bind to specific effectormolecules are referred to as receptors. In the present invention,antibodies are preferred receptors. Antibodies can be used to targetliposomes to specific cell-surface ligands. For example, certainantigens expressed specifically on tumor cells, referred totumor-associated antigens (TAAs), may be exploited for the purpose oftargeting mcl-1 antibody-containing liposomes directly to the malignanttumor. Since the mcl-1 gene product may be indiscriminate with respectto cell type in its action, a targeted delivery system offers asignificant improvement over randomly injecting non-specific liposomes.Preferably, the target tissue is ovarian and the target cell is agranulosa cell. A number of procedures can be used to covalently attacheither polyclonal or monoclonal antibodies to a liposome bilayer.Antibody-targeted liposomes can include monoclonal or polyclonalantibodies or fragments thereof such as Fab, or F(ab′)₂, as long as theybind efficiently to an the antigenic epitope on the target cells.Liposomes may also be targeted to cells expressing receptors forhormones or other serum factors.

The antibodies and substantially purified mcl-1 polypeptide of thepresent invention are ideally suited for the preparation of a kit. Sucha kit may comprise a carrier means being compartmentalized to receive acarrier means being compartmentalized to receive in close confinementtherewith one or more container means such as vials, tubes and the like,each of said container means comprising the separate elements of theassay to be used.

The types of assays which can be incorporated in kit form are many, andinclude, for example, competitive and non-competitive assays. Typicalexamples of assays which can utilize the antibodies of the invention areradioimmunoassays (RIA), enzyme immunoassays (EIA), enzyme-linkedimmunosorbent assays (ELISA), and immunometric, or sandwichimmunoassays.

The term “immunometric assay” or “sandwich immunoassay”, includessimultaneous sandwich, forward sandwich and reverse sandwichimmunoassays. These terms are well understood by those skilled in theart. Those of skill will also appreciate that antibodies according tothe present invention will be useful in other variations and forms ofassays which are presently known or which may be developed in thefuture. These are intended to be included within the scope of thepresent invention.

In performing the assays it may be desirable to include certain“blockers” in the incubation medium (usually added with the labeledsoluble antibody). The “blockers” are added to assure that non-specificproteins, proteases, or anti-heterophilic immunoglobulins to anti-mcl-1immunoglobulins present in the experimental sample do not cross-link ordestroy the antibodies on the solid phase support, or the radiolabeledindicator antibody, to yield false positive or false negative results.The selection of “blockers” therefore may add substantially to thespecificity of the assays described in the present invention.

It has been found that a number of nonrelevant (i.e., nonspecific)antibodies of the same class or subclass (isotype) as those used in theassays (e.g., IgG1, IgG2a, IgM, etc.) can be used as “blocker”. Theconcentration of the “blockers” (normally 1-100 μg/μl) is important, inorder to maintain the proper sensitivity yet inhibit any unwantedinterference by mutually occurring cross reactive proteins in thespecimen.

In addition to the polynucleotides of the invention, the monoclonalantibodies of the invention can also be used, alone or in combinationwith effector cells (Douillard, et al., Hybridoma, 5 Supp.1:S139, 1986),for immunotherapy in an animal having a cell proliferative disorderwhich expresses mcl-1 polypeptide with epitopes reactive with themonoclonal antibodies of the invention.

When used for immunotherapy, the monoclonal antibodies of the inventionmay be unlabeled or labeled with a therapeutic agent. These agents canbe coupled either directly or indirectly to the monoclonal antibodies ofthe invention. One example of indirect coupling is by use of a spacermoiety. These spacer moieties, in turn, can be either insoluble orsoluble (Diener, et al., Science, 231:148, 1986) and can be selected toenable drug release from the monoclonal antibody molecule at the targetsite. Examples of therapeutic agents which can be coupled to themonoclonal antibodies of the invention for immunotherapy are drugs,radioisotopes, lectins, and toxins.

The drugs which can be conjugated to the monoclonal antibodies of theinvention include non-proteinaceous as well as proteinaceous drugs. Theterms “non-proteinaceous drugs” encompasses compounds which areclassically referred to as drugs, for example, mitomycin C,daunorubicin, and vinblastine.

The proteinaceous drugs with which the monoclonal antibodies of theinvention can be labeled include immunomodulators and other biologicalresponse modifiers. The term “biological response modifiers” encompassessubstances which are involved in modifying the immune response in suchmanner as to enhance the destruction of an mcl-1-associated tumor forwhich the monoclonal antibodies of the invention are specific. Examplesof immune response modifiers include such compounds as lymphokines.Lymphokines include tumor necrosis factor, the interleukins,lymphotoxin, macrophage activating factor, migration inhibition factor,colony stimulating factor, and interferon. Interferons with which themonoclonal antibodies of the invention can be labeled includealpha-interferon, beta-interferon and gamma-interferon and theirsubtypes.

In using radioisotopically conjugated monoclonal antibodies of theinvention for immunotherapy certain isotypes may be more preferable thanothers depending on such factors as tumor cell distribution as well asisotope stability and emission. If desired, the tumor cell distributioncan be evaluated by the in vivo diagnostic techniques described above.Depending on the cell proliferative disease some emitters may bepreferable to others. In general, alpha and beta particles emittingradioisotopes are preferred in immunotherapy. For example, if an animalhas solid tumor foci a high energy beta emitter capable of penetratingseveral millimeters of tissue, such as ⁹⁰Y, may be preferable. On theother hand, if the cell proliferative disorder consists of simple targetcells, as in the case of leukemia, a short range, high energy alphaemitter, such as ²¹²Bi, may be preferable. Examples of radioisotopeswhich can be bound to the monoclonal antibodies of the invention fortherapeutic purposes are ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁶⁷Cu, ²¹²Bi, ²¹¹At, ²¹²Pb,⁴⁷Sc, ¹⁰⁹Pd, and ¹⁸⁸Re.

Lectins are proteins, usually isolated from plant material, which bindto specific sugar moieties. Many lectins are also able to agglutinatecells and stimulate lymphocytes. However, ricin is a toxic lectin whichhas been used immunotherapeutically. This is preferably accomplished bybinding the alpha-peptide chain of ricin, which is responsible fortoxicity, to the antibody molecule to enable site specific delivery ofthe toxic effect.

Toxins are poisonous substances produced by plants, animals, ormicroorganisms, that, in sufficient dose, are often lethal. Diphtheriatoxin is a substance produced by Corynebacterium diphtheria which can beused therapeutically. The toxin consists of an alpha and beta subunitwhich under proper conditions can be separated. The toxic A componentcan be bound to an antibody and used for site specific delivery to amcl-1 bearing cell for which the monoclonal antibodies of the inventionare specific. Other therapeutic agents which can be coupled to themonoclonal antibodies of the invention are known, or can be easilyascertained, by those of ordinary skill in the art.

The labeled or unlabeled monoclonal antibodies of the invention can alsobe used in combination with therapeutic agents such as those describedabove. Especially preferred are therapeutic combinations comprising themonoclonal antibody of the invention and immunomodulators and otherbiological response modifiers.

Thus, for example, the monoclonal antibodies of the invention can beused in combination with alpha-interferon. This treatment modalityenhances monoclonal antibody targeting of carcinomas by increasing theexpression of monoclonal antibody reactive antigen by the carcinomacells (Greiner, et al., Science, 235:895, 1987). Alternatively, themonoclonal antibody of the invention could be used, for example, incombination with gamma-interferon to thereby activate and increase theexpression of Fc receptors by effector cells which, in turn, results inan enhanced binding of the monoclonal antibody to the effector cell andkilling of target tumor cells. Those of skill in the art will be able toselect from the various biological response modifiers to create adesired effector function which enhances the efficacy of the monoclonalantibody of the invention.

When the monoclonal antibody of the invention is used in combinationwith various therapeutic agents, such as those described herein, theadministration of the monoclonal antibody and the therapeutic agentusually occurs substantially contemporaneously. The term “substantiallycontemporaneously” means that the monoclonal antibody and thetherapeutic agent are administered reasonably close together withrespect to time. Usually, it is preferred to administer the therapeuticagent before the monoclonal antibody. For example, the therapeutic agentcan be administered 1 to 6 days before the monoclonal antibody. Theadministration of the therapeutic agent can be daily, or at any otherinterval, depending upon such factors, for example, as the nature of thetumor, the condition of the patient and half-life of the agent.

Using monoclonal antibodies of the invention, it is possible to designtherapies combining all of the characteristics described herein. Forexample, in a given situation it may be desirable to administer atherapeutic agent, or agents, prior to the administration of themonoclonal antibodies of the invention in combination with effectorcells and the same, or different, therapeutic agent or agents. Forexample, it may be desirable to treat patients with leukemia or lymphomaby first administering gamma-interferon and interleukin-2 daily for 3 to5 days, and on day 5 administer the monoclonal antibody of the inventionin combination with effector cells as well as gamma-interferon, andinterleukin-2.

It is also possible to utilize liposomes with the monoclonal antibodiesof the invention in their membrane to specifically deliver the liposometo the area of the tumor expressing mcl-1. These liposomes can beproduced such that they contain, in addition to the monoclonal antibody,such immunotherapeutic agents as those described above which would thenbe released at the tumor site (Wolff, et al., Biochemical et BiophysicalActa, 802:259, 1984).

The dosage ranges for the administration of monoclonal antibodies of theinvention are those large enough to produce the desired effect in whichthe symptoms of the malignant disease are ameliorated. The dosage shouldnot be so large as to cause adverse side effects, such as unwantedcross-reactions, anaphylactic reactions, and the like. Generally, thedosage will vary with the age, condition, sex and extent of the diseasein the patient and can be determined by one of skill in the art. Thedosage can be adjusted by the individual physician in the event of anycomplication. Dosage can vary from about 0.1 mg/kg to about 2000 mg/kg,preferably about 0.1 mg/kg to about 500 mg/kg, in one or more doseadministrations daily, for one or several days. Generally, when themonoclonal antibodies of the invention are administered conjugated withtherapeutic agents, lower dosages, comparable to those used for in vivodiagnostic imaging, can be used.

The monoclonal antibodies of the invention can be administeredparenterally by injection or by gradual perfusion over time. Themonoclonal antibodies of the invention can be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously,intracavity, or transdermally, alone or in combination with effectorcells.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present such as, forexample, antimicrobials, anti-oxidants, chelating agents and inert gasesand the like.

The invention also relates to a method for preparing a medicament orpharmaceutical composition comprising the polynucleotides or themonoclonal antibodies of the invention, the medicament being used fortherapy of mcl-1 associated cell proliferative disorders.

The invention also provides a method of preventing programmed cell death(apoptosis) in a cell comprising introducing into the cell, functionalmcl-1 polypeptide or an expression vector containing an mcl-1 encodingpolynucleotide sequence. For example, this method can be used toincrease the viability of the cell in cell culture during an ex vivoprotocol or for long term in vitro cell propagation. Similarly,introduction of mcl-1 polypeptide or an expression vector containing themcl-1 encoding polynucleotide sequence into a cell can be utilized as ameans for inducing differentiation in a cell capable of undergoingdifferentiation.

The following example are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLE 1 Construction and Screening of TPA Induced ML-1 Cell cDNALibrary

To identify “early-induction” genes, poly(A)+ RNA was isolated from ML-1cells induced with TPA for three hours. A complementary DNA (cDNA)library was constructed and was screened by differential hybridization,using probes derived from the TPA-induced cells AND uninduced controls.A cDNA clone representing mcl-1 was identified based upon preferentialhybridization to the probe from induced cells.

ML-1 cells were programmed to differentiate with TPA as describedpreviously (K. M. Kozopas, H. L. Buchan, R. W. Craig, J. Cell Physiol.,145, 575 (1990). After preincubation under reduced serum conditions for3 days, cells were exposed to 5×10⁻¹⁰ M TPA for 3 hours. Poly(A)+ RNAfrom these TPA-induced cells was used for oligo(dT)-primed first strandcDNA synthesis, which was carried out with Moloney murine leukemia virusreverse transcriptase (Bethesda Research Laboratories, Gaithersburg,Md.). After second strand cDNA synthesis, double stranded cDNA of >500basepairs was cloned into the EcoRI site of lambda gt10. The libraryobtained was subjected to differential screening , using ³²P-labeledcDNA probes synthesized by reverse transcription of poly(A)⁺ RNA fromthe TPA-induced cells and a parallel culture of uninduced cells. A cloneexhibiting preferential hybridization to the probe from induced cells(clone dif8C, containing nucleotides 3150-3946 of mcl-1) was isolatedand subcloned into the Bluescript plasmid (Stratagene, La Jolla,Calif.). This clone was used to rescreen the cDNA library to obtainclones spanning the mcl-1 cDNA. Clones spanning the mcl-1 coding regionwere also obtained from a cDNA library from TPA-induced U-937 cells(Clontech, Palo Alto, Calif.). Sequencing was carried out using theSequenase enzyme (U.S. Biochemicals, Cleveland, Ohio).

EXAMPLE 2 Time Course of Expression of mcl-1

The time course of expression of mcl-1 was monitored during thedifferentiation of ML-1 cells. ML-1 cells were exposed to 5×10⁻¹⁰ M TPAand assayed at various times for expression of mcl-1 and other mRNAs(Panels A, B) and for cell surface markers of differentiation (Panel C).Panel A shows expression of mcl-1 as determined by Northern blotting.Probes for mcl-1 (dif8C-p3.2, see FIG. 3), beta-actin, myb (pCM8), andCD11b were hybridized to total RNA from cells exposed to TPA for theindicated times [in hours (h) or days (d)]. Panel B shows the timecourse of expression of mcl-1. Autoradiographs such as the one shown in(A) were subjected to densitometric scanning. The values for expressionof mcl-1 were normalized by dividing by the corresponding value forbeta-actin, which did not change with time. Relative expression of mcl-1was estimated as the ratio of expression in TPA-induced cells to that inuninduced controls. Panel C shows the time course of appearance of cellsurface markers of differentiation. Flow cytometry (FACSCAN) wasperformed using phycoerythrin-conjugated antibodies to CD11b and CD14(Becton Dickenson, Mountain View, Calif.). Background fluorescence,determined using isotype matched control antibodies, was subtracted. Thepercentage of morphologically differentiating cells averaged 40%, 82%,and 90% in cultured induced with TPA for 1, 2, and 3 days, respectively,and 3.5% in uninduced control cultures, as found previously. Thesedifferentiating cells were predominantly immature forms on day 1, withapproximately equal numbers of immature and mature forms present on days2 and 3. Cell growth in the TPA-induced culture was decreased by about93%, as found previously. Each point represents the average ±SE of 2-5experiments.

While expression of mcl-1 was low in uninduced cells, it increaseddramatically early in induction with TPA (FIG. 1A). This increase wasseen within one hour and was maximal (>6-fold at 3 hours) (FIG. 1B). Atthis time, the programming of differentiation was in progress andexpression of c-myb mRNA was decreased (Craig, et al., Ca. Res., 44:442,1984), although no changes in morphology or differentiation markers hadoccurred (FIGS. 1A, C). These markers did not begin to appear until16-24 hours (FIG. 1C), when expression of mcl-1 was in decline [to <50%of maximum (FIGS. 1A, B)]. Expression of mcl-1 also increased early inthe TPA-induced differentiation of other myeloid leukemia cell lines,including HL-60, and U-937. The rapid up-regulation and down-regulationof this “early-induction” gene prior to phenotypic differentiation isthus reminiscent of the pattern of expression of the “early-response”genes important in proliferation (Nathans, et al., Cold Spring HarborSymposia on Quantitative Biology L111, pp. 893-900, 1988).

The genes in the mcl-1/bcl-2 family exhibit intriguing parallels intheir patterns of expression. mcl-1 was isolated from ML-1 cells, whichare derived from a patient who developed acute myeloid leukemia afterthe remission of a T-cell lymphoma; bcl-2 was originally identified inpatients with follicular B-cell lymphoma. TPA elicited an early increasein expression of mcl-1 (FIG. 1), and can combine with other agents tocause similar increases in bcl-3 and BHRF1. Expression of mcl-1 isincreased early in myeloid cells programmed to differentiate and stopproliferating without dying (FIG. 1). Expression of bcl-2 is increasedin lymphoid cells programmed to remain viable and selected for furtherdifferentiation. Expression of BHRF1 is increased early in the lyticcycle of the virus and early in serum-induced stimulation ofproliferation. Genes i the mcl-1/bcl-2 family are thus characterized,not only by homology in the carboxyl region/hydrophobic tail (FIG. 4),but also by the fact that changes in expression may occur as an earlyevent in the programs that determine call proliferation,differentiation, and/or viability.

It is not yet known how these parallels in patterns of expression mighttranslate into parallels in function. bcl-2 has a role in themaintenance of viability through inhibition of programmed cell death; itappears to operate in a variety of cells, including hematopoietic celllines deprived of required growth factors, certain types of B-cells(e.g., B-memory cells), and T-cells under specific circumstances. Theidentification of mcl-1 allows it to be tested for a similar role in themaintenance of viability, apparently operating in myeloid cells duringthe induction of differentiation. bcl-2 is distinct from many oncogenesand growth-factor related genes in that it can enhance viability withoutstimulation proliferation; the viable cells remain in G₀/G₁ phase of thecell cycle. mcl-1 may also play a role in the accumulation in G₀/G₁ thataccompanies differentiation. Deregulation of bcl-2 is thought tocontribute to tumorigenesis by increasing cell survival, therebyincreasing the probability of accumulation of additional changes (suchas rearrangements of the c-myc oncogene). The discovery of the relatedmcl-1 gene leads to the identification of a growing number of geneswhich affect the programming of cell death and/or differentiation. Thesegenes may prove to be as important, in tumorigenesis and its reversal,as the wide variety of known families of oncogene and tumor suppressorgenes.

EXAMPLE 3 Sequence of mcl-1

A panel of overlapping mcl-1 cDNA clones was initially obtained. Theseclones defined a sequence of 3,946 basepairs, in accord with the longesttranscript size of 3.8 kb (FIGS. 5a and 5 b). The longest open readingframe within this sequence is preceded by a Kozak sequence (Kozak, Nucl.Acids Res., 12:857, 1984) and an upstream in-frame stop codon. Severalpolymorphisms exist in the nucleotide sequence. When nucleotide 740 isC, amino acid 227 is alanine (A); when nucleotide 740 is T, amino acid227 is valine (V). Using this reading frame, the mcl-1-encoded protein(FIG. 2A) was predicted to contain 350 amino acids and to have amolecular size of 37.3 kD. FIG. 2 shows the deduced amino acid sequenceof the mcl-1 protein and schematic representation of the cDNA. In panelA, PEST sequences are underlined and asterisks indicate pairs ofarginines. The arrow indicates the region with homology to bcl-2 anddouble lines indicate the hydrophobic carboxyl tail. Plus signs indicatepositively charge flanking amino acid resides. Amino acid residue 227was valine in clones from ML-1 and alanine in those from U-937. Aminoacid residue 1 corresponds to nucleotides 61-63 of the cDNA. Panel Bshows a schematic representation of mcl-1. The boxed area represents theprotein coding region; this is followed by a line representing the3′-untranslated region (discontinuous line). The amino terminus of mcl-1has some characteristics of a signal sequence (as does that of BHRF1) ,but does not function as such in in vitro translation in the presence ofmicrosomal membranes.

Parallels within this family continue downstream of the protein codingregion: Both mcl-1 and bcl-2 have long 3′-untranslated regions [2.8 kbin mcl-1 (FIG. 2B)]. Both have multiple potential polyadenylation sitesand mRNA destabilization signals. The presence of severalpolyadenylation sites in mcl-1 (FIG. 2B) might relate to the twotranscripts observed (FIG. 1A). The presence of mRNA destabilizationsignals might relate to the transience of the increase in expression(FIGS. 1A, B). Translocations involving bcl-2 frequently occur in the340 -untranslated region, often within the “major breakpoint region”(mbr) of about 150 nucleotides. Interestingly, the 3′-untranslatedregion of mcl-1 contains a stretch with sequence similarity to this mbr(FIG. 2B).

The size of mcl-1 encoded protein was confirmed by in vitro translationusing mcl-1 cDNAs from two independent sources (FIG. 3, lanes 1-2). FIG.3 in vitro translation of mcl-1 mRNA. A cDNA lacking the firstmethionine yielded a truncated protein of the size predicted from thesecond methionine (FIG. 3, lane 3). Plasmids representing mcl-1 werelinearized at the 3′ end of the cDNA and used to prepare mRNA by invitro transcription with T7 polymerase (Pharmacia, Piscataway, N.J.).This mRNA was translated in vitro in the presence of ³⁵S-methionine(1000 Ci/mmol, Amersham, Arlington Heights, Ill.), using a rabbitreticulocyte lysate system (Novagen, Madison, Wis.). The reactionproducts were separately by sodium dodecyl sulfate polyacrylamide(12.5%) gel electrophoresis and detected by autoradiography. Lane 1shows reaction products from a cDNA containing the complete mcl-1 codingsequence (clone dif8C-1A6, containing nucleotides 52 to 1484). Lane 2shows reaction product s from a different cDNA clone (clone dif8C-3.2,containing nucleotides 7 to 1484). Lane 3 shows reaction products from acDNA clone lacking the methionine at amino acid residue 1 (clonedif8C-7C, containing nucleotides 278 to 1484). Clones dif8C-1A6 anddif8C-7C were from the cDNA library from U-937 cells; clone dif8C-3.2was from the cDNA library from ML-1 cells. Lane 4 shows no mRNA and lane5 shows the molecular weight markers. (Traces of the marker are alsopresent in lane 4).

In its amino terminal portion, the predicted mcl-1 protein containsseveral interesting features, including two “PEST” sequences (Rogers, etal., Science, 234:364, 1986), enriched in proline (P), glutamic acid(E), serine (S), and threonine (T) and four pairs of arginines (FIGS. 2,2A, B). These sequences are present in a variety of oncoproteins andother proteins that undergo rapid turn-over. Their presence in mcl-1suggests that this protein might be expected to be expressed, like themRNA (FIG. 1), primarily in the early stages of differentiation.Interestingly, bcl-2 does not have PEST sequences, although it doesdemonstrate differentiation-stage specific expression (e.g., in myeloidcells and intestinal epithelium, where expression declines duringmaturation).

It is in the carboxyl region that mcl-1 has sequence homology to bcl-2[35% amino acid identity and 59% similarity in 139 amino acid residues,FIG. 2A, B (arrows) and FIG. 4]. FIG. 4 shows the alignment of thecarboxyl portions of mcl-1, bcl-2, and BHRF1. The BESTFIT program (GCGSequence Analysis Software) was used to align the amino acid sequencesof the carboxyl portions of mcl-1, bcl-2alpha [human (Tsujimoto, et al.,Proc. Natl. Acad. Sci. USA, 83:5214, 1986)] and BHRF 1 [Epstein-Barrvirus (Pearson et al., Virology, 160:151, 1987)], gaps being inserted tomaximize overlap. The symbols used are: |=amino acid identity; :=aminoacid comparison value ≧0.5; .=amino acid comparison value ≧0.1. Boldletters indicate residues that are identical in the three proteins.Double lines flanked by plus signs indicate the hydrophobic carboxyltail. Asterisks indicate areas of high conservation; a consensussequence for mcl-1 and bcl-2 is shown at the top, where conservednon-identical residues are indicated as follows: a=P, A, G, S, T; i=L,I, V, M; f=F, Y, W; d=Q, N, E, D; h=H, K, R, as determined by theSIMPLIFY program. Differences in reported sequences of bcl-2 are inunderlined italics. Differences between human and mouse (Negrini, etal., Cell, 49:455, 1987) bcl-2 are double underlined.

bcl-2 was identified in follicular B-cell lymphomas, the majority ofwhich have a specific translocation involving chromosomes 14 and 18.This translocation juxtaposes bcl-2 with the immunoglobulin heavy chainlocus and results in deregulated expression of an unaltered bcl-2 geneproduct. bcl-2 has not been found to have homology to previouslydescribed cellular oncogenes or to contain motifs characteristic ofother known gene families. The carboxyl region of bcl-2 is known toexhibit some homology to the BHRF1 gene from Epstein-Barr virus (25%),and this parallels the fact that the carboxyl regions of human and mousebcl-2 exhibit greater identity (98% in 144 amino acid residues) than dothe amino terminal portions (76%). Thus, the discovery of mcl-1 providesthe first example of a cellular gene with homology to bcl-2 and suggeststhe existence of a unique gene family represented by mcl-1, bcl-2, andBHRF-1. Homology in the carboxyl region appears to be an importantdefining characteristic of this family.

At their extreme carboxyl termini, mcl-1, bcl-2 (bcl-2alpha), and BHRF1each contain a potential membrane spanning domain (20 hydrophobic aminoacid residues indicated with double lines and flanked by positivelycharged residues in FIGS. 2A and 4). This hydrophobic carboxyl tail isknown to mediate the membrane-association of bcl-2, which has recentlybeen localized to mitochondrial membranes (Hockenberg, et al., Nature,348:334, 1990). BHRF-1 is also membrane-associated. The finding of ahydrophobic carboxyl tail in mcl-1 suggests that the potential formembrane association may be another important characteristic of genes inthis family.

The foregoing is meant to illustrate, but not to limit, the scope of theinvention. Indeed, those of ordinary skill in the art can readilyenvision and produce further embodiments, based on the teachings herein,without undue experimentation.

4 3946 base pairs nucleic acid single linear DNA (genomic) unknown mcl-1CDS 61..1110 /note= “When nucleotide 740 = C, amino acid 227 = A; whennucleotide 740 = T, amino acid 227 = V.” 1 TCCAGTAAGG AGTCGGGGTCTTCCCCAGTT TTCTCAGCCA GGCGGCGGCG GCGACTGGCA 60 ATGTTTGGCC TCAAAAGAAACGCGGTAATC GGACTCAACC TCTACTGTGG GGGGGCCGGC 120 TTGGGGGCCG GCAGCGGCGGCGCCACCCGC CCGGGAGGGC GACTTTTGGC TACGGAGAAG 180 GAGGCCTCGG CCCGGCGAGAGATAGGGGGA GGGGAGGCCG GCGCGGTGAT TGGCGGAAGC 240 GCCGGCGCAA GCCCCCCGTCCACCCTCACG CCAGACTCCC GGAGGGTCGC GCGGCCGCCG 300 CCCATTGGCG CCGAGGTCCCCGACGTCACC GCGACCCCCG CGAGGCTGCT TTTCTTCGCG 360 CCCACCCGCC GCGCGGCGCCGCTTGAGGAG ATGGAAGCCC CGGCCGCTGA CGCCATCATG 420 TCGCCCGAAG AGGAGCTGGACGGGTACGAG CCGGAGCCTC TCGGGAAGCG GCCGGCTGTC 480 CTGCCGCTGC TGGAGTTGGTCGGGGAATCT GGTAATAACA CCAGTACGGA CGGGTCACTA 540 CCCTCGACGC CGCCGCCAGCAGAGGAGGAG GAGGACGAGT TGTACCGGCA GTCGCTGGAG 600 ATTATCTCTC GGTACCTTCGGGAGCAGGCC ACCGGCGCCA AGGACACAAA GCCAATGGGC 660 AGGTCTGGGG CCACCAGCAGGAAGGCGCTG GAGACCTTAC GACGGGTTGG GGATGGCGTG 720 CAGCGCAACC ACGAGACGGTCTTCCAAGGC ATGCTTCGGA AACTGGACAT CAAAAACGAA 780 GACGATGTGA AATCGTTGTCTCGAGTGATG ATCCATGTTT TCAGCGACGG CGTAACAAAC 840 TGGGGCAGGA TTGTGACTCTCATTTCTTTT GGTGCCTTTG TGGCTAAACA CTTGAAGACC 900 ATAAACCAAG AAAGCTGCATCGAACCATTA GCAGAAAGTA TCACAGACGT TCTCGTAAGG 960 ACAAAACGGG ACTGGCTAGTTAAACAAAGA GGCTGGGATG GGTTTGTGGA GTTCTTCCAT 1020 GTAGAGGACC TAGAAGGTGGCATCAGGAAT GTGCTGCTGG CTTTTGCAGG TGTTGCTGGA 1080 GTAGGAGCTG GTTTGGCATATCTAATAAGA TAGCCTTACT GTAAGTGCAA TAGTTGACTT 1140 TTAACCAACC ACCACCACCACCAAAACCAG TTTATGCAGT TGGACTCCAA GCTGTAACTT 1200 CCTAGAGTTG CACCCTAGCAACCTAGCCAG AAAAGCAAGT GGCAAGAGGA TTATGGCTAA 1260 CAAGAATAAA TACATGGGAAGAGTGCTCCC CATTGATTGA AGAGTCACTG TCTGAAAGAA 1320 GCAAAGTTCA GTTTCAGCAACAAACAAACT TTGTTTGGGA AGCTATGGAG GAGGACTTTT 1380 AGATTTAGTG AAGATGGTAGGGTGGAAAGA CTTAATTTCC TTGTTGAGAA CAGGAAAGTG 1440 GCCAGTAGCC AGGCAAGTCATAGAATTGAT TACCCGCCGA ATTCATTAAT TTACTGTAGT 1500 AGTGTTAAGA GAAGCACTAAGAATGCCAGT GACCTGTGTA AAAGTTACAA GTAATAGAAC 1560 TATGACTGTA AGCCTCAGTACTGTACAAGG GAAGCTTTTC CTCTCTCTAA TTAGCTTTCC 1620 CAGTATACTT CTTAGAAAGTCCAAGTGTTC AGGACTTTTA TACCTGTTAT ACTTTGGCTT 1680 GGTTCCATGA TTCTTACTTTATTAGCCTAG TTTATCACCA ATAATACTTG ACGGAAGGCT 1740 CAGTAATTAG TTATGAATATGGATATCCTC AATTCTTAAG ACAGCTTGTA AATGTATTTG 1800 TAAAAATTGT ATATATTTTTACAGAAAGTC TATTTCTTTG AAACGAAGGA AGTATCGAAT 1860 TTACATTAGT TTTTTTCATACCCTTTTGAA CTTTGCAACT TCCGTAATTA GGAACCTGTT 1920 TCTTACAGCT TTTCTATGCTAAACTTTGTT CTGTTCAGTT CTAGAGTGTA TACAGAACGA 1980 ATTGATGTGT AACTGTATGCAGACTGGTTG TAGTGGAACA AATCTGATAA CTATGCAGGT 2040 TTAAATTTTC TTATCTGATTTTGGTAAGTA TTCCTTAGAT AGGTTTTCTT TGAAAACCTG 2100 GGATTGAGAG GTTGATGAATGGAAATTCTT TCACTTCATT ATATGCAAGT TTTCAATAAT 2160 TAGGTCTAAG TGGAGTTTTAAGGTTACTGA TGACTTACAA ATAATGGGCT CTGATTGGGC 2220 AATACTCATT TGAGTTCCTTCCATTTGACC TAATTTAACT GGTGAAATTT AAAGTGAATT 2280 CATGGGCTCA TCTTTAAAGCTTTTACTAAA AGATTTTCAG CTGAATGGAA CTCATTAGCT 2340 GTGTGCATAT AAAAAGATCACATCAGGTGG ATGGAGAGAC ATTTGATCCC TTGTTTGCTT 2400 AATAAATTAT AAAATGATGGCTTGGAAAAG CAGGCTAGTC TAACCATGGT GCTATTATTA 2460 GGCTTGCTTG TTACACACACAGGTCTAAGC CTAGTATGTC AATAAAGCAA ATACTTACTG 2520 TTTTGTTTCT ATTAATGATTCCCAAACCTT GTTGCAAGTT TTTGCATTGG CATCTTTGGA 2580 TTTCAGTCTT GATGTTTGTTCTATCAGACT TAACCTTTTA TTTCCTGTCC TTCCTTGAAA 2640 TTGCTGATTG TTCTGCTCCCTCTACAGATA TTTATATCAA TTCCTACAGC TTTCCCCTGC 2700 CATCCCTGAA CTCTTTCTAGCCCTTTTAGA TTTTGGCACT GTGAAACCCC TGCTGGAAAC 2760 CTGAGTGACC CTCCCTCCCCACCAAGAGTC CACAGACCTT TCATCTTTCA CGAACTTGAT 2820 CCTGTTAGCA GGTGGTAATACCATGGGTGC TGTGACACTA ACAGTCATTG AGAGGTGGGA 2880 GGAAGTCCCT TTTCCTTGGACTGGTATCTT TTCAACTATT GTTTTATCCT GTCTTTGGGG 2940 GCAATGTGTC AAAAGTCCCCTCAGGAATTT TCAGAGGAAA GAACATTTTA TGAGGCTTTC 3000 TCTAAAGTTT CCTTTGTATAGGAGTATGCT CACTTAAATT TACAGAAAGA GGTGAGCTGT 3060 GTTAAACCTC AGAGTTTAAAAGCTACTGAT AAACTGAAGA AAGTGTCTAT ATTGGAACTA 3120 GGGTCATTTG AAAGCTTCAGTCTCGGAACA TGACCTTTAG TCTGTGGACT CCATTTAAAA 3180 ATAGGTATGA ATAAGATGACTAAGAATGTA ATGGGGAAGA ACTGCCCTGC CTGCCCATCT 3240 CAGAGCCATA AGGTCATCTTTGCTAGAGCT ATTTTTACCT ATGTATTTAT CGTTCTTGAT 3300 CATAAGCCGC TTATTTATATCATGTATCTC TAAGGACCTA AAAGCACTTT ATGTAGTTTT 3360 TAATTAATCT TAAGATCTGGTTACGGTAAC TAAAAGCCTG TCTGCCAAAT CCAGTGGAAA 3420 CAAGTGCATA GATGTGAATTGGTTTTTAGG GGCCCCACTT CCCAATTCAT TAGGTATGAC 3480 TGTGGAAATA CAGACAAGGACTTAGTTGAT ATTTTGGGCT TGGGGCAGTG AGGGCTTAGG 3540 ACACCCCAAG TGGTTTGGGAAAGGAGGAGG GAGTGGTGGG TTTATAGGGG AGGAGGAGGC 3600 AGGTGGTCTA AGTGCTGACTGGCTACGTAG TTCGGGCAAA TCCTCCAAAA GGGAAAGGGA 3660 GGATTTGCTT AGAAGGATGGGGCTCCCAGT GACTACTTTT TGACTTCTGT TTGTCTTACG 3720 CTTCTCTCAG GGAAAAACATGCAGTCCTCT AGTGTTTCAT GTACATTCTG TGGGGGGTGA 3780 ACACCTTGGT TCTGGTTAAACAGCTGTACT TTTGATAGCT GTGCCAGGAA GGGTTAGGAC 3840 CAACTACAAA TTAATGTTGGTTGTGCAAAT GTAGTGTGTT TCCCTAACTT TCTGTTTTTC 3900 CTGAGAAAAA AAAATAAATCTTTTATTCAA ATAAAAAAAA AAAAAA 3946 350 amino acids amino acid singlelinear peptide unknown mcl-1 CDS 1..3946 /note= “When nucleotide 740 =C, amino acid 227 = A; when nucleotide 740 = T, amino acid 227 = V”Peptide 1..350 /note= “When nucleotide 740 = C, amino acid 227 = A; whennucleotide 740 = T, amino acid 227 = V.” 2 Met Phe Gly Leu Lys Arg AsnAla Val Ile Gly Leu Asn Leu Tyr Cys 1 5 10 15 Gly Gly Ala Gly Leu GlyAla Gly Ser Gly Gly Ala Thr Arg Pro Gly 20 25 30 Gly Arg Leu Leu Ala ThrGlu Lys Glu Ala Ser Ala Arg Arg Glu Ile 35 40 45 Gly Gly Gly Glu Ala GlyAla Val Ile Gly Gly Ser Ala Gly Ala Ser 50 55 60 Pro Pro Ser Thr Leu ThrPro Asp Ser Arg Arg Val Ala Arg Pro Pro 65 70 75 80 Pro Ile Gly Ala GluVal Pro Asp Val Thr Ala Thr Pro Ala Arg Leu 85 90 95 Leu Phe Phe Ala ProThr Arg Arg Ala Ala Pro Leu Glu Glu Met Glu 100 105 110 Ala Pro Ala AlaAsp Ala Ile Met Ser Pro Glu Glu Glu Leu Asp Gly 115 120 125 Tyr Glu ProGlu Pro Leu Gly Lys Arg Pro Ala Val Leu Pro Leu Leu 130 135 140 Glu LeuVal Gly Glu Ser Gly Asn Asn Thr Ser Thr Asp Gly Ser Leu 145 150 155 160Pro Ser Thr Pro Pro Pro Ala Glu Glu Glu Glu Asp Glu Leu Tyr Arg 165 170175 Gln Ser Leu Glu Ile Ile Ser Arg Tyr Leu Arg Glu Gln Ala Thr Gly 180185 190 Ala Lys Asp Thr Lys Pro Met Gly Arg Ser Gly Ala Thr Ser Arg Lys195 200 205 Ala Leu Glu Thr Leu Arg Arg Val Gly Asp Gly Val Gln Arg AsnHis 210 215 220 Glu Thr Val Phe Gln Gly Met Leu Arg Lys Leu Asp Ile LysAsn Glu 225 230 235 240 Asp Asp Val Lys Ser Leu Ser Arg Val Met Ile HisVal Phe Ser Asp 245 250 255 Gly Val Thr Asn Trp Gly Arg Ile Val Thr LeuIle Ser Phe Gly Ala 260 265 270 Phe Val Ala Lys His Leu Lys Thr Ile AsnGln Glu Ser Cys Ile Glu 275 280 285 Pro Leu Ala Glu Ser Ile Thr Asp ValLeu Val Arg Thr Lys Arg Asp 290 295 300 Trp Leu Val Lys Gln Arg Gly TrpAsp Gly Phe Val Glu Phe Phe His 305 310 315 320 Val Glu Asp Leu Glu GlyGly Ile Arg Asn Val Leu Leu Ala Phe Ala 325 330 335 Gly Val Ala Gly ValGly Ala Gly Leu Ala Tyr Leu Ile Arg 340 345 350 154 amino acids aminoacid single linear protein unknown bcl-2alpha Protein 1..154 3 Leu SerPro Val Pro Pro Val Val His Leu Thr Leu Arg Gln Ala Gly 1 5 10 15 AspAsp Phe Ser Arg Arg Tyr Arg Arg Asp Phe Ala Glu Met Ser Arg 20 25 30 GlnLeu His Leu Thr Pro Phe Thr Ala Arg Gly Arg Phe Ala Thr Val 35 40 45 ValGlu Glu Leu Phe Arg Asp Gly Val Asn Trp Gly Arg Ile Val Ala 50 55 60 PhePhe Glu Phe Gly Gly Val Met Cys Val Glu Ser Val Asn Arg Glu 65 70 75 80Met Ser Pro Leu Val Asp Asn Ile Ala Leu Trp Met Thr Glu Tyr Leu 85 90 95Asn Arg His Leu His Thr Trp Ile Gln Asp Asn Gly Gly Trp Asp Ala 100 105110 Phe Val Glu Leu Tyr Gly Pro Ser Met Arg Pro Leu Phe Asp Phe Ser 115120 125 Trp Leu Ser Leu Lys Thr Leu Leu Ser Leu Ala Leu Val Gly Ala Cys130 135 140 Ile Thr Leu Gly Ala Tyr Leu Gly His Lys 145 150 152 aminoacids amino acid single linear protein unknown BHRF-1 Protein 1..152 4Leu Ser Pro Glu Asp Thr Val Val Leu Arg Tyr His Val Leu Leu Glu 1 5 1015 Glu Ile Ile Glu Arg Asn Ser Glu Thr Phe Thr Glu Thr Trp Asn Arg 20 2530 Phe Ile Thr His Thr Glu His Val Asp Leu Asp Phe Asn Ser Val Phe 35 4045 Leu Glu Ile Phe His Arg Gly Asp Pro Ser Leu Gly Arg Ala Leu Ala 50 5560 Trp Met Ala Trp Cys Met His Ala Cys Arg Thr Leu Cys Cys Asn Gln 65 7075 80 Ser Thr Pro Tyr Tyr Val Val Asp Leu Ser Val Arg Gly Met Leu Glu 8590 95 Ala Ser Glu Gly Leu Asp Gly Trp Ile His Gln Gln Gly Gly Trp Ser100 105 110 Thr Leu Ile Glu Asp Asn Ile Pro Gly Ser Arg Arg Phe Ser TrpThr 115 120 125 Leu Phe Leu Ala Gly Leu Thr Leu Ser Leu Leu Val Ile CysSer Tyr 130 135 140 Leu Phe Ile Ser Arg Gly Arg His 145 150

What is claimed is:
 1. A method for identifying a cell expressing mcl-1polypeptide comprising contacting the cell with a reagent which binds tothe polypeptide, and detecting binding of the reagent, therebyidentifying a cell expressing mcl-1.
 2. The method of claim 1, whereinthe reagent is a probe.
 3. The method of claim 2, wherein the probe isan antibody.
 4. The method of claim 3, wherein the antibody ispolyclonal.
 5. The method of claim 3, wherein the antibody ismonoclonal.
 6. The method of claim 1, wherein the cell is ahematopoietic cell.
 7. The method of claim 2, wherein the probe isdetectably labeled.
 8. The method of claim 7, wherein the label isselected from the group consisting of a radioisotope, a bioluminescentcompound, a chemiluminescent compound, a fluorescent compound, a metalchelate, or an enzyme.